DNA transposons are mobile elements that can move from one position in a genome to another. Naturally, transposons play roles in evolution as a result of their movements within and between genomes. Geneticists have used transposons as tools for both gene delivery and insertional mutagenesis or gene tagging in lower animals (Shapiro, Genomics, 1992; 86:99-111) but not, until recently, in vertebrates. Transposons are relatively simple genetic systems, consisting of some genetic sequence bounded by inverted terminal repeats and a transposase enzyme that acts to cut the transposon out of one source of DNA and paste it into another DNA sequence (Plasterk, Cell, 1993; 74:781-786). Autonomous transposons carry the transposase gene inside the transposon whereas non-autonomous transposons require another source of transposase for their mobilization. Among the DNA transposable elements, members of Tc1/mariner family have been found in a wide variety of organisms, ranging from fungi to humans (Doak et al., Proc. Natl. Acad. Sci. USA, 1994; 91:942-946; Radice et al., Mol. Gen. Genet., 1994; 244:606-612). Both the Tc1 and mariner transposons can be transposed using purified transposase protein (Lampe et al., EMBO J., 1996; 15:5470-5479; Vos et al., Genes Dev., 1996; 10:755-761; Tosi et al., Nucl. Acids Res., 2000; 28:784-790). This simplicity in mechanism and broad range of invasion suggested that such a transposon would be useful to develop into a vertebrate transformation vector. However, following an intensive search in vertebrates, primarily fish, not a single active Tc1/mariner-type transposon was found (Izsvák et al., Mol. Gen. Genet., 1995; 247:312-322; Ivics et al., Proc. Natl. Acad. Sci. USA, 1996; 93:5008-5013). Of the nearly 10,000 Tc1/mariner-type transposons found in the haploid human genome, none appear to have active transposase genes (Lander et al., Nature, 2001; 409:860-921; Venter et al., Science, 2001; 291:1304-1351).
Accordingly, a functional Tc1-like transposon system was reconstructed from sequences found in salmonid fish. The synthetic transposase was named Sleeping Beauty (SB), owing to its restoration to activity from a transposon that lost its activity more than 10 million years ago (Ivics et al., Cell, 1997; 91:501-510). The SB transposon appears to obey a cardinal rule of Tc1/mariner transposons, it integrates only into a TA-dinucleotide sequence, which is duplicated upon insertion in the host genome (Ivics et al., Cell, 1997; 91:501-510; Luo et al., Proc. Natl. Acad. Sci. USA, 1998; 95:10769-10773). Transposons in the Tc1/mariner superfamily can be sorted into three groups based on the different length of inverted terminal repeats (ITRs) and the different numbers and patterns of transposase-binding sites in the ITRs (Plasterk et al., Trends Genet., 1999; 15:326-332). One group of transposons has a structure that suggests that there are direct repeats (DRs) within the ITRs or inverted repeat (IR) sequences that have accumulated mutations over time. These are referred to as the IR/DR group, whose members have a pair of binding-sites containing short, 15-20 bp DRs at the ends of each IR, which are about 200-250 bp in length. SB transposons were placed in this group (Ivics et al., Proc. Natl. Acad. Sci. USA, 1996; 93:5008-5013; Ivics et al., Cell, 1997; 91:501-510). Both binding sites are essential for transposition-deletion or mutation of either DR or ITR virtually abolishes transposition (Ivics et al., Cell, 1997; 91:501-510; Izsvák et al., J. Mol. Biol., 2000; 302:93-102).
The SB system is functional in a wide range of vertebrate cells, from fish to humans (Plasterk et al., Trends Genet., 1999; 15:326-332; Izsvák et al., J. Mol. Biol., 2000; 302:93-102). It has been used to deliver genes for long-term gene expression in mice (Yant et al., Nature Genet., 2000; 25:35-40; Fischer et al., Proc. Nail Acad. Sci. USA, 2001; 98:6759-6764; Dupuy et al., Genesis, 2001; 30:82-88; Dupuy et al., Proc. Natl. Acad. Sci. USA, 2002; 99:4495-4499; Horie et al., Proc. Natl. Acad. Sci. USA, 2001; 98:9191-9196) and zebrafish. The SB system is nearly 10-fold more efficient than other Tc1/mariner-type transposons in human cells (Fischer et al., Proc. Natl. Acad. Sci. USA, 2001; 98:6759-6764), although the efficiency drops off as the size of the transposon increases (Izsvák et al., J. Mol. Biol., 2000; 302:93-102; Karsi et al., Mar. Biotechnol., 2001; 3:241-245). These findings suggest that the SB system has considerable promise as a tool for transgenesis and insertional mutagenesis in vertebrates as well as gene therapy in humans.
For such applications, highly active transposons are required. Early results suggested that the SB system in mice might be extremely low because mobilization was extremely infrequent in ES cells (Luo et al., Proc. Natl. Acad. Sci. USA, 1998; 95:10769-10773). However more recent results involving remobilization of SB transposons suggest that the system may be a useful tool in mammals (Yant et al., Nature Genet., 2000; 25:35-40; Fischer et al., Proc. Natl. Acad. Sci. USA, 2001; 98:6759-6764; Dupuy et al., Genesis, 2001; 30:82-88; Dupuy et al., Proc. Natl. Acad. Sci. USA, 2002; 99:4495-4499; Horie et al., Proc. Natl. Acad. Sci. USA, 2001; 98:9191-9196).